【作者】 谢启应；

【导师】 孙明；

【作者基本信息】 中南大学 ， 内科学， 2003， 博士

【摘要】 目的：高血压致靶器官损伤可归结于两种血管病变：动脉硬化和动脉粥样硬化(atherosclerosis，AS)。临床和动物实验均证实，合并高血压时AS的发生和发展明显加速，但其分子机制不详。目前发现，高血压促进AS发生发展除了血流动力学的直接作用外，尚有其他复杂的非血流动力学因素的作用。而这对防治高血压致AS血管病变可能具有更重要的意义。最近研究认为AS是一种慢性炎症免疫性疾病。有多种细胞如单核细胞、内皮细胞、平滑肌细胞等在多种免疫分子如粘附因子、趋化因子等协调下相互作用，相互影响，参与了AS的发生发展。单核细胞趋化因子-1(monocyte chemoattractant protein-1，MCP-1)是一种主要作用于单核细胞/巨噬细胞的强效趋化剂，诸多实验证实在AS的早期发生和发展中起着重要的作用。血管紧张素Ⅱ(angiotensin Ⅱ，AngⅡ)除了致血压增高的作用外，近年研究认为它还可直接刺激内皮细胞、平滑肌细胞和单核细胞等表达炎症因子如MCP-1、白细胞介素-6(IL-6)等。因此AngⅡ可能是高血压和AS的联系纽带，通过刺激炎症因子的表达在高血压促AS形成过程中起重要作用。本文拟从临床和实验研究着手，以MCP-1为指标，初步探讨高血压时MCP-1表达可能机制及其临床意义。为进一步探讨高血压促AS发生发展的分子机制提供理论和临床依据。 方法：从临床研究、动物实验和细胞培养三方面：(1)采用双抗体夹心法(ELISA)和放射免疫法测定32例1、2级低中危险度单纯中南大学博士学位论文 中文摘要高血压患者和 30例正常健康者外周血中 MCP刁和 Aug 11的浓度变化，并探讨循环 MCPl浓度与 And、血压之间的关系；＊）构建两肾一夹u kidneyl elm，ZKIC）高血压大鼠模型，观察其急性期主动脉MCPlmRNA的表达以及氯沙坦治疗对其的影响，采用原位杂交和RT一PCR方法；（3）探讨 Aug 11对人脐静脉内皮细胞（HUVECs）MCP一 1表达的影响，采用ELISA和RT－PCR方法。 结果：二．临床研究：门）同正常对照组相比，高血压患者外周血MCP－l的浓度明显增高（8．66土2．55 VS 15．45上1．13，P＜0．001），而Angll 浓度两组无差异（32．98土14．61 Vs32．51上10．65，P＝0．887）； （）外周血 MCPl浓度同循环中的 Aug 11浓度、血压升高的程度无相关性（R＝0．013，－0．082，－0．01 respec亡ively，P＞0．05）。2．动物实验：门）正常血压大鼠主动脉壁未见 MCP刁 的表达；ZKIC高血压大鼠在急性期主动脉壁即有表达，且随着时间的延长，其表达进一步增加；氯沙坦干预可明显减少其表达① 值分别为0、0．58土0．10、1．14土0．09、1．52土0．ZO、O．66士0．07）；（2）急性期ZKIC 高血压大鼠主动脉壁主要是在血管内膜表达 MCP习帕NA。o乃 高血压大鼠主动脉壁的 Aug 11浓度与 MCP刁mRNA表达强度有相同的增加趋势。3．－细胞培养：0）不同浓度的 Aug 11刺激 HUVEC试验发现，未刺激组 HUVEC可见极少量 MCP－lmRNA的表达，而 An＊育迅速诱导其表达，在 Ans11浓度在 IX 10－’mol／1时其表达最强（随浓度增加 OD值分别为0．40士0．08、0．95土0．21、1．16土0．13、1．48士0．24、1．01土0．18）2，（2）用 An二刺激 2 ，J’时时 MCP－1。RNA的表达即明显增加。在 4小 11 歹－中南大学博士学位论文 中文摘要时时表达最强烈，而24小时时己基本恢复（随时间增加，各组OD值分别为0．310．08、0．99士0．21．1．19土0．30、1．03土0．18、0．8土0．15和 0．34土0．07）；（3）随着Angll 刺激时间的延长，MCP一1的浓度明显增加（20．24士 0．36 VS 25．26士 0．28，P＜0．00）：（4）预先加入氯沙坦可阻断 Aug 11的上述作用（CTL、Aug 11． Los、Los＋Aug 11四组的OD值分别为0．38上0．09，二．10士0．27，0．36上0．08，0．38士0．18，P＜O．O二）。 结论：1．高血压有较高的AS发生率可能与MCPl异常表达有关，后者可能在高血压致动脉粥样硬化性靶器官损伤过程中起作用。2．在刺激组织、细胞表达MCPl中起主要作用的可能是组织局部的 Ans 11而非循环中的 Ans 11。3．Ans 11刺激组织细胞肌P－1表达可能主要由AT；受体介导。更多还原

【Abstract】 Background and Objective: The target-organ damage due to hypertension is attributed to two different pathological process: arteriosclerosis and atherosclerosis (AS). As is all known, hypertension is an important risk factor for atherosclerosis, however, the mechanism responsible for the atherosclerotic lesion formation accelerated by hypertension is still unclear. The extension of the lesion is not only related to the force of hemodynamics, but also due to other non-hemodynamic factors. It has been commonly recognized that AS is a chronic inflammatory disease. Monocyte chemoattract protein-1 (MCP-1), a powerful chemotactic factor, has been identified to play an important pathophysiological role in early atherogenesis. Angiotensin II (Ang II), a neurohormonal regulatory factor for arterial blood pressure, has been proved to be able to affect the expression of enormous inflammatory factors such as MCP-1,IL-6 by stimulating endothelia, vascular smooth muscle cells (VSMC) and monocytes. So, Ang II may play an important role in the development of atherosclerotic lesion in hypertension, and it has been conjectured to be the link of hypertension and atherosclerosis. In the present study, we investigated the MCP-1 levels in hypertensive subjects and in aorta of 2K1C hypertensive rats, and the effect of Ang II on the expression of MCP-1 in human umbilical vein endothelial cells (HUVECs).Methods: Three parts were performed sequentially: (1) the fasting serum levels of MCP-1 and Ang II were measured in 32IVhypertensive and 30 healthy subjects by ELISA and radio-immunoassay respectively. (2) 41 rats including normotensive and 2K1C hypertensive rats were divided into 5 groups: negative control group, positive central groups of 7 days> 14 days^ 28 days and treatment group. The expression of MCP-lmRNA in rat aorta was detected by RT-PCR and in situ hybridization respectively. The levels of Ang II in serum and in aorta were measured by radioimmunoassay. (3) The effects of Ang II of different concentrations and stages on the expression of MCP-1 were performed in HUVECs; The MCP-1 protein in supernatant was assayed by ELISA, and MCP-lmRNA expression was detected by RT-PCR.Results: (1) The concentration of MCP-1 in hypertensions were significantly higher than that of normotensives (18.66?.55 Vs 15.45?.13,P<0.001), but the concentration of Ang II had no significant difference between the two groups (32.98?4.61 Vs 32.51?0.65, P=0.887); and no correlation was found between MCP-1 and Ang II or MCP-1 and blood pressure (R=0.013,-0.082,-0.011 respectively,P>0.05); (2) No expression of MCP-lmRNA was detected by RT-PCR and in situ hybridization in aorta of normotensive rats; after 7 days, The expression of MCP-lmRNA was detected in aorta of 2K1C rats ^specially in intima , and it significantly increased with a duration-dependent mode, with a peak at 28 days. After treatment with losartan (25mg/kg/d) for 28 days, the expression of MCP-lmRNA decreased significantly compared with nontreatment groups(MCP-l/GAPDH ratio: 0, 0.58?.10> 1.14?.09> 1.52?.20 > 0.66?.07 respective, PO.01). (3) A little expression MCP-lmRNA in HUVECs was detected without existence of Ang II, however, Ang II dramatically activated the expression of MCP-lmRNA.after preincubation for 6 hours at a dose-dependent manner. The strongest expression of MCP-lmRNA was at the dose of lxlO~7mol/l (MCP-1/GAPDHratio: 0.40?.08> 0.95?.2K 1.16?.13> 1.48?.24> 1.01?.18 respective). (4)with incubation of HUVECs with Ang II for 2 hours, the expression of MCP-lmRNA was markedly increased, and the strongest expression was found at the fourth hour, but almost recovered to basal level at the 24th hour(MCP-l/GAPDH ratio: 0. 3?.08> 0.99?.2K 1.19?.30> 1.03?.18> 0.8?.15 and 0.34?.07 respective).(5)The concentration of MCP-1 in supernatant of HUVECs increased gradually after 12 and 24 hours when HUVECs were stimulated by Ang II (20.24?.36 Vs 25.26?.28, PO.001), (6) Losartan had no effect on the expression of MCP-1 in HUVECs. But the effect o更多还原